Non-bile acid cholephils such as bilirubin and sulfobromophthalein (BSP), free fatty acids (FFA), and bile acids represent 3 distinct classes of organic anions efficiently taken up by the liver despite tight albumin binding. Organic anion uptake is, in fact, a major hepatic function, which may be impaired in disease. This proposal seeks to clarify the role in carrier mediated organic anion uptake of BSP/bilirubin binding protein (BSP/BR-BP) and hepatic plasma membrane fatty acid binding protein (h- FABP/pm), two proteins in the basolateral plasma membrane of the hepatocyte with high affinities for BSP/bilirubin and FFA, respectively, identified and isolated during years 1-11 of this project. Specifically, we will complete the physicochemical characterization of the proteins including aminoacid sequencing; confirm their transport function in liposome reconstitution studies; prepare polyclonal and monoclonal antibodies against each; complete ongoing development of immunoassays to quantitate cell surface expression of both proteins; and correlate changes in the uptake velocities of radiolabeled BSP/bilirubin and FFA produced by phenobarbital or clofibrate administration, or by fibroblast to adipocyte differentiation in 3T3-L1 cells, with changes in the cell surface expression of the corresponding proteins. Additionally, we will use the antibodies to screen a rat liver cDNA library in bacteriophage lambda-gt11; clone the cDNA inserts from positive isolates into an appropriate plasmid and use them screen for full length cDNAs; restriction map and sequence the inserts/cDNAs and use these data to search for homologies between these proteins and with other known proteins; radiolabel the inserts with [32P] and determine the tissue distribution and response to pharmacologic manipulation of the relevant mRNA's by slot blot, and the size and number of the corresponding messages in the liver by Northern hybridization; and estimate the sizes of the relevant genes by Southern hybridization. We will further confirm protein function by using the cloned cDNAs in genetic reconstitution studies in, e.g., Xenopus oocytes, and COS cells. Finally, the inserts will be used to screen genomic libraries. A long term goal is to study the promoter/enhancer regions of the genes for phenobarbital responsive and clofibrate responsive (FFA binding) hepatic proteins, to determine whether the apparently coordinated response to each agent is mediated by common regulatory sequences within each of the responsive genes.